The present invention relates to double mutants of bacteriorhodopsin, wherein the amino-acid positions 85 and 96 of the opsin of a wild-type strain of Halobacteria are mutated.
2. The Prior Art
Bacteriorhodopsin is a photoactivatable proton pump which occurs in Halobacteria such as, for example, Halobacterium halobium, and is composed of a protein portion (bacterioopsin) and of a chromophore (retinal) (see, for example, Oesterhelt and Tittor, TIBS 14 (1989), pp. 57-61). Native bacteriorhodopsin, which is obtainable from the wild type of Halobacterium, has an absorption maximum at a wavelength of 570 nm.
The absorption maximum of the intermediate in the excited state has a wavelength of 412 nm. Bacteriorhodopsin is organized in two-dimensional crystalline regions of the cell membrane, called the purple membrane. Up to 80 percent of the cell membrane of Halobacterium halobium may be composed of this purple membrane. Isolation of the bacteriorhodopsin in the form of this purple membrane is possible by known means (Oesterhelt and Stoeckenius, Methods Enzymol. 31 Biomembranes, pp. 667-678, 1974).
Besides bacteriorhodopsin, Halobacteria produce halorhodopsin, a light-driven chloride, bromide and iodide pump. The structure of the latter closely resembles that of bacteriorhodopsin, but it is not produced in cell membrane structures resembling the purple membrane. The content of halorhodopsin in the cell membrane is only one tenth of that of bacteriorhodopsin. The isolation of this halorhodopsin is significantly more elaborate because of these properties. Like bacteriorhodopsin, halorhodopsin has a photocycle, and the absorption maximum of the ground state is at a wavelength of 578 nm. The intermediate with the longest life has an absorption maximum of 520 nm.
Because of the optoelectrical properties of bacteriorhodopsin and other rhodopsins, attempts are being made to use these substances for biocomputers and for pattern recognition (Biosystems 19 (1986), pp. 223-236). It is desirable, for these purposes, to shift the absorption maximum of bacteriorhodopsin and of the intermediate in the photocycle to longer wavelengths in order to be able to replace the costly special lasers which are required for the excitation of the wild-type bacteriorhodopsin by low-cost commercially available lasers.
The property of halorhodopsin of being able, with the aid of light energy, to pump chloride ions would be interesting for an application for the desalination of solutions. To date, the isolation of halorhodopsin has been so elaborate that no economic utilization has been possible. It is, therefore, desirable to be able to isolate rhodopsins with the properties of halorhodopsin in the form of purple membranes.
To date, several mutants of bacteriorhodopsin which have shown an effect of the mutation on the photocycle and the absorption maxima have been disclosed.
Mogi et al (PNAS USA 85, 4148-152, 1988) describe single amino-acid mutants in which the amino acid Asp in positions 85, 96, 115 and 212 is replaced by Glu or Asn. In other mutants, Asp in position 212 is replaced by Asn, Glu or Ala. The proteins were expressed in E. coli. The proteins were purified from E. coli. Bacteriorhodopsin-like chromophores were regenerated by detergent, phospholipid treatment and addition of retinal. It was shown that proton translocation was completely abolished by replacement of Asp-85 with Asn and partly (&lt;10%) by replacement of Asp-96 by Asn and Asp-212 by Glu. Mutation of Asp-85 to Asn resulted in a shift in the absorption maximum to .lambda..sub.max =590 nm.
Soppa et al [J. Biol. Chem., 264, 22, 13049-13056, (1989)] describes bacteriorhodopsin muteins with the following single amino-acid mutations Asp-85 to Glu; Asp-96 to Asn; Asp-96 to Gly. These muteins were generated by random mutagenesis with X-ray or UV radiation and selected by bromodeoxyuracil selection of photosynthetically negative phenotypes. Thus, they were not generated in a directed manner. These muteins differed from the modified proteins investigated by Mogi et al in that they were expressed in Halobacteria and isolated therefrom in structures resembling purple membranes. Mutation of Asp-96 slowed down the kinetics of the photocycle. No alteration in the spectral properties of bacteriorhodopsin occurred in this case. Replacement of amino acid Asp-85 by Glu results in a shift in the absorption maximum from 568 nm to 610 nm. Deprotonation of amino acid Glu-85 shifts the absorption maximum to about 530 nm.
Subramaniam et al [Proc. Natl. Acad. Sci USA, 87, pp. 1013-1017, (1990) investigated the change in the absorption spectrum of bacteriorhodopsin by mutations at position Asp-85 and Arg-82. Replacement of Asp-85 with Asn yields a blue chromophore (.lambda..sub.max =588 nm). Proton translocation is no longer detectable. No investigations of the photocycle were carried out.